SUMMARY: The long-term goal of Project 1 is to identify mechanisms responsible for more efficient genome and epigenome maintenance in long-lived animal species. Genome instability, including genomic rearrangements arising from errors during repair of DNA breaks, and dysregulation of epigenetic landscapes are believed to be contributing causes of aging. In the current period of support, we demonstrated that the efficiency of DNA double-strand break (DSB) repair, but not nucleotide excision repair (NER), correlates positively with maximum lifespan. Furthermore, we demonstrated that SIRT6 protein, which serves as an upstream regulator of DSB repair, is a major factor responsible for the differences in DSB repair efficiency between species. SIRT6 ability to promote DSB repair correlated strongly with maximum lifespan. By dissecting SIRT6 sequence variation across rodents with large differences in lifespan, we identified five amino acids fully responsible for the differences in SIRT6 activity in promoting DSB repair, as well as the differences in SIRT6 deacetylation and mono-ADP-ribosylation activities. Other preliminary studies identified SIRT6 as a key factor in epigenome stability during aging. We found that SIRT6 suppresses activation of transposable elements in aged mice, restores inducibility of NRF2 target genes, and confers more youthful expression signatures to senescent cells. Based on these findings, our objectives are to expand our analysis of SIRT6 changes responsible for improved DSB repair to other long-lived species; test whether long-lived species have more stable epigenome maintenance upon DNA damage and characterize the role of SIRT6 in this process; and finally, test whether the identified five amino acid changes that improve mouse SIRT6 to the level found in long-lived species result in lifespan extension. Our overarching hypothesis is that longevity is associated with improved DSB repair and epigenome maintenance and stimulation of SIRT6 activity can improve these processes and extend the lifespan. We propose to: (1) Integrate data from multiple long-lived species to determine the landscape of amino acid changes in SIRT6 that are associated with longevity and improved DSB repair; (2) Test whether the ability to recover epigenome organization following DNA damage correlates with maximum lifespan, and identify the role of SIRT6 in this process. We will collaborate with Project 2 to test the effect of hyaluronan on epigenome maintenance, and with Project 3 to compare genome and epigenome maintenance in different rodent species upon DNA damage. (3) Construct a ?beaverized? SIRT6 mouse containing five beaver amino acid substitutions responsible for enhanced SIRT6 activity and with Core C test whether these mice show improved health and lifespan. We will work with Project 3 to characterize genome and epigenome stability in these mice and with Project 4 to determine whether improved DNA repair in these mice results in more youthful metabolism and methylation age. The proposed research will identify targets for interventions aimed at promoting genome and epigenome stability and extending lifespan.